Interactions between cytoplasmic myosin and actin are responsible for a variety of cellular motile activities. As in muscle contraction, hydrolysis of ATP by myosin provides the required energy. While the actin-activated ATPases of vertebrate smooth muscle and nonmuscle myosins are regulated by phosphorylation of their 20,000-Da light chains, the mechanism of this regulation varies from one type of cell to another. Unphosphorylated gizzard myosin appears to be inactive, and phosphorylation causes a large increase in its actin-activated ATPase. This myosin has frequently been used as a model for all vertebrate smooth and nonmuscle myosins. However, we have found that phosphorylation regulates the actin-activated ATPase of calf thymus cytoplasmic myosin not by increasing the maximum rate of ATP hydrolysis but rather by increasing its affinity for actin. At low actin concentrations, phosphorylation stimulates the ATPase activity of thymus myosin, but at high actin concentrations or in the presence of tropomyosin, unphosphorylated and phosphorylated thymus myosin have comparable activities. We have subsequently found that the mechanism of activation of a mammalian smooth muscle myosin isolated from calf aorta is more like that of thymus myosin than that of gizzard myosin. This mammalian smooth muscle myosin has significant actin-activated ATPase activity in the absence of light chain phosphorylation. We have used skeletal muscle myofibrils from which the myosin has been extracted to demonstrate that unphosphorylated thymus and aorta myosins, but not unphosphorylated gizzard myosin, can cause contraction. The ability of unphosphorylated aorta myosin to cause contraction is consistent with the maintenance of tension by mammalian smooth muscles when light chain phosphorylation has returned to resting levels. These results also suggest that mammalian cells contain some mechanism in addition to light chain phosphorylation for regulating actomyosin based motility.